The complete DNA sequences of two biologically very important control regions have not yet been elucidated. One is a promotor region (the start of mRNA synthesis) and the other is a replication origin (the start of DNA synthesis). The objective of this proposal is to determine the DNA sequence around the replication origin at each of the three replicative stages during the life cycle of phages phi X174 and M13. The origin of Replicative Stage I (ss DNA yields RF DNA) will be determined by filling in with DNA polymerase I and radioactive triphosphates, the gap formed when M13 (plus) strand is incubated with a soluble enzyme extract from pol I-cells. In the presence of manganese, DNA polymerase I can incorporate one ribotriphosphate and the other three deoxyribo-triphosphates to give bonds which are susceptible to alkali. These specific cleavages give small fragments which can be sequenced and overlapped to give the full sequence. The origin of Replicative Stage II (RF DNA yields RF DNA) will be determined by sequencing the nucleotide region around the single unique nick formed by gene A protein in the (plus) strand of phi X174 RF DNA. The DNA sequence recognized in Replicative Stage III (RF DNA yield ss DNA) will be determined by analyzing the sequence at the termini of linear phi X174 (plus) strands found in phage phi X174 derived from t.s. ligase mutants of E. coli. M13 and phi X174 are unrelated bacteriophages. M13 replication appears to resemble certain characteristics of "plasmid" replication while phi X174 replication resembles "chromosomal" replication. Since DNA from both bacteriophages will be used, it will be possible to compare the DNA sequences of these unrelated phages which are recognized by the replication apparatus of the same host, E. coli. This project will lay the groundwork for future projects involving sequence analysis of replication origins in the DNA of eucaryotic organisms. BIBLIOGRAPHIC REFERENCES: Miller, D.M., Gubbins, E.J., Pegg, E. and Donelson, J. E. (1976) Biochemistry (in press). Transcription and Translation of cloned Drosophila DNA Fragments in E. coli.